Type 2 diabetes affects 366 million people globally, and 25.8 million in the US alone. It is one of the major public health and economic burdens of the 21st century. Despite the scale of the problem our fundamental understanding of the disease itself is incomplete. In patients with type 2 diabetes, exercise reduces insulin resistance in muscle and increases glucose sensitivity in the pancreas. The objective of this R21 application is to explore the cellular and molecular links between skeletal muscle contraction and downstream regulation of pancreatic insulin secretion. Our central hypothesis is that contracting skeletal muscle secretes myokines that target ?-cells and participate in the regulation of glucose stimulated insulin secretion. To test this hypothesis we will generate primary cell cultures from vastus lateralis muscle of newly diagnosed patients with type 2 diabetes and non-diabetic age and gender matched controls, and measure the myokine secretome response to contraction using electrical pulse stimulation (EPS). We will utilize state-of-the-art proteomic analyses, including 2D electrophoresis and mass spectrometry to identify non-targeted myokines, and immunoassays to identify a targeted myokine signature in the media from muscle cells that undergo contraction. We will use computational filtering to identify the secreted myokines. Global physiological validation of our findings will be assessed from the collective effect of these myokines on glucose stimulated insulin secretion from isolated human pancreatic islets. Based on our preliminary human studies in type 2 diabetes, insulin secretion was increased after exercise training. We expect that glucose stimulated insulin secretion and stimulus-secretory- coupling will be increased in islets incubated in media from EPS-conditioned cells. It is expected that myokines secreted during EPS mediate increased pancreatic insulin secretion. We will use chemical inhibition, siRNA and immunocytochemistry to explore the endoplasmic reticulum-Golgi network as a likely secretory pathway for contraction-induced myokine secretion. These studies will provide the initial steps towards a comprehensive and complementary analysis of a biochemical link between skeletal muscle contraction and downstream pancreatic insulin secretion in a clinically relevant population of individuals with type 2 diabetes. We expect that these proof-of-principle studies will lead to the identification of specific myokines that can be used for drug development and diabetes management. The discoveries will also further our understanding of skeletal muscle as an endocrine organ and its role in the regulation of glucose metabolism and insulin secretion in type 2 diabetes.